Enhancers are regions of the genome that are able to regulate gene expression from a distance, and, unlike the genes they regulate, enhancers are highly specific to certain types of cells or conditions. This cell-type-specificity and context-dependence of enhancers has raised the possibility that we might be able to target enhancers in order to modulate gene expression within a particular subtype of cells. Such a targeted modulation of gene expression, called enhancer therapy, would be valuable in treating tissue-specific autoimmune diseases such as type 1 diabetes, where T cells destroy the beta cells in the pancreas. In such cases, total immune suppression- as with Cyclosporin A- is a blanket response, but enhancer therapy might allow us to limit the effect of treatment to the subset of pathogenic cells. For example, we might target enhancers specific to T cells attacking the beta cells to block inflammatory genes in only those cells. However, analysis of enhancers through high-throughput sequencing has revealed that many enhancers are redundantly wired, forming a robust regulatory system. Increasing or decreasing the activity of any single enhancer often has minimal effects on its target gene or genes. The efficiency of enhancer therapy therefore lies in the identification of keystone enhancers-enhancers that have an outsized effect on the genes they regulate. This project aims to address this question of keystone enhancers first by investigating a cellular context in which enhancer therapy would be beneficial: T cells subjected to high- and low-affinity stimulation. We will generate an enhancer atlas for these subgroups of T cells using both traditional immunological assays and high-throughput sequencing methods, thereby giving us insight into what happens when T cells are stimulated with antigens of differing affinity. Secondly, we will build software and a computational framework for identifying candidate keystone enhancers from multiple integrated high-throughput data sets. With the successful completion of this project, we will be positioned to validate candidate keystone enhancers using genome-editing techniques. Thus, this project represents a significant step in the progression of enhancer therapy from theory to reality, and holds substantial promise for the treatment of autoimmune diseases.